The sense of taste is essential for the discrimination between nutrients and non-nutrients through the activation of taste receptors in the oral cavity. Bitter taste has evolved as a warning signal by alerting the body to potentially harmful substances. Multiple bitter taste receptors (T2Rs) are expressed in gut epithelial cells and they are likely to serve as transducers for the detection of luminal contents. Intraluminal T2R ligands activate vagal afferents, induce release of gut hormones, and modulate food intake and gastrointestinal functions. Interestingly, in the mouse colon, a subset of T2Rs, which are predominantly localized to enteroendocrine (EEC) cells, and the major T2R signaling molecule, a-gustducin, are upregulated by high fat diet, which is known to induce dysbiosis of the gut microbiota, inflammation, obesity, and metabolic disorders. T2R subtypes are also upregulated in the colon of overweight/obese humans, where they are only localized to EEC cells. Moreover, the quorum-sensing molecule, N-3- (oxododecanoyl)-homoserine lactone (AHL) activates a GPCR cascade associated with bitter taste transduction in the EEC cell line, STC 1, and induce release of gut peptides from intestinal mucosa through a pathway likely involving T2Rs. Quorum-sensing molecules are critical for bacteria communication and they modulate host immune response. This proposal will test the hypothesis that intestinal T2Rs mediate host functional responses to changes in the gut microbiota by sensing bacterial products and inducing release of gut hormones. Specific Aim 1 will determine whether alterations of gut microbiota composition regulate T2Rs expression and whether blockade of T2R signaling affects the host response induced by high-fat diet using a-gustducin KO mice. Specific Aim 2 will test whether bacteria-produced quorum sensing molecules activate gut T2Rs by comparing the effects of AHL and T2R ligands on intracellular Ca2+ and ERK phosphorylation in STC 1 cells and dissociated mucosal cells, and on CCK, GLP-1 and -2, and PYY release from intestinal mucosa of normal mice or mice with a-gustducin gene deletion. These studies will use morphological, imaging and signaling approaches with in vivo animal models of high fat diet, manipulations of the gut microbiota, and germ free mice, and in vitro model systems including STC 1 cells, isolated mucosal cells, and organotypic cultures of the intestinal mucosa. Mouse lines null for a-gustducin or with the a-gustducin promoter driving GFP will also be used. siRNA knockdown of T2Rs or a-gustducin, inhibitors of signaling pathways, and a T2R inhibitor will be used to control for specificity of cellular responses. The homeostasis between host and microbiota in the gut lumen is an important factor in diseases such as obesity and metabolic disorders. The impact of this proposal is that a greater understanding of the roles of T2Rs in the detection of intraluminal bacteria and mediation of host response, will provide novel targets for the treatment of obesity and other disorders involving the microbiome.